The Induction Of Proteins ESAT-6 And Ag85B Secreted By Mycobacterium Tuberculosis On FoxP3mRNA In Peripheral Blood

Background and Objective:Tuberculosis (TB) is one of the leading infectious diseases, which is a respiratory disease endangering people's health seriously. In our previous study,we confirmed that the level of CD4+CD25+ regulatory T cells (Treg) in patients with drug resistance-cavernous pulmonary tuberculosis significantly decreased after the effective surgical treatment. There existed a high correlation between increased level of CD4+CD25+ Treg and Mycobacterium tuberculosis(MTB) infection. However, the mechanism of the changes of the quantity of CD4+CD25+Treg of TB patients is indefinite. So we treated human peripheral blood with ESAT-6 and Ag85B, and then detected the CD4+CD25 +T cells (%PBMC) in peripheral blood by flow cytometry. However, due to the activation of T cells(including CD4+CD25- T cells), CD25 also up-regulated. As a result, CD25 was not considered as a specific marker of Treg. Forkhead-like transcription factor FoxP3 is a specific biomarker of the CD4+CD25+Treg cells. FoxP3mRNA expressed in Treg is quantitative, but did not express in activated CD4+CD25- T cells. The cellular level of CD4+CD25+Treg cells was positively correlated with the level of the FoxP3mRNA. In order to detect the level of Treg more precisely, we tested the level of FoxP3mRNA with Real-Time PCR (RT-PCR). By detecting the level of FoxP3mRNA, we could observe the level of CD4+CD25+FoxP3+Treg cells in peripheral blood after treated with the ESAT-6 and Ag85B, conforming the conclusions got before.Materials and Methods:1. From the patients in The Third People's Hospital of Shantou, we obtained the results of sputum culture, drug sensitive test and general information, describing the distribution of local TB.2. Volunteers were selected randomly from healthy people, the latent infester, drug-resistant TB patients, drew their blood.3. Processed blood with ESAT-6, Ag85B, ESAT-6+Ag85B, BCG in different concentrations(0μg/ml, 1μg/ml, 2μg/ml, 4μg/ml), cultured for different hours(24h, 48h, 72h), then observed the level of FoxP3mRNA by using RT-PCR.Results: 1. In epidemiological surveys, we found men significantly outnumber women with positive sputum culture and men were older than women. The drug-resistant TB had a high proportion in drug sensitive test.2. Treated the peripheral blood with the ESAT-6 and Ag85B in different concentrations and cultured for different time, we observed the increasing concentration of the proteins could induce FoxP3mRNA expression up-regulation. In addition, the level of FoxP3mRNA in peripheral blood reached peak when cultured for 24h, then declined slightly with time. All of these had not statistically significant.3. Comparing the three groups (healthy control group, latent infectious group and the patients) with no stimulation, we found the mRNA level of FoxP3 between the patients and latent infectious group were higher than that of the control group.4. Before and after processing with the proteins, the mRNA level of FoxP3 after treatment was higher than that before the treatment (P<0.05).5. Comparing the three groups after treated with the different proteins, we found that the mRNA level of FoxP3 of the infectious group was higher than the healthy control and the patients, and the patients is lower then the healthy control.Conclusions: The drug-resistant TB is severe currently. There is an urgent need to explore the mechanism of immune escape of MTB, helping to choose the best treatment. Stimulated the peripheral blood with antigens and cultured in vitro, we found the FoxP3mRNA expression had increased significantly after treated with ESAT-6 and Ag85B, indicating that there was a dose-response effect between the concentrations of the proteins and the mRNA level of FoxP3. FoxP3 was the main protein secreted by CD4+CD25+Treg, which meant that the amount of bacteria increased can induce FoxP3mRNA to up-regulate. CD4+CD25+ FoxP3+Treg cells were also the indispensable element inducing the M. tuberculosis to evade immune clearance, so we could draw the conclusion that ESAT-6 and Ag85B have a connection with the immune escape of MTB.